Method for the identification of the clonal source of a restriction fragment

ABSTRACT

The present invention relates to a high throughput method for the identification and detection of molecular markers wherein restriction fragments are generated and suitable adaptors comprising (sample-specific) identifiers are ligated. The adapter-ligated restriction fragments may be selectively amplified with adaptor compatible primers carrying selective nucleotides at their 3′ end. The amplified adapter-ligated restriction fragments are, at least partly, sequenced using high throughput sequencing methods and the sequence parts of the restriction fragments together with the sample-specific identifiers serve as molecular markers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.13/344,162, which is a divisional of U.S. patent application Ser. No.12/373,220 filed Mar. 11, 2011, Issued as U.S. Pat. No. 8,178,300 on May15, 2012, which is a U.S. National Stage of PCT/NL2007/000177, filedJul. 10, 2001, which claims the benefit of U.S. Provisional ApplicationNo. 60/830,121, filed Jul. 12, 2006, all of which are incorporatedherein by reference in entirety.

FIELD OF THE INVENTION

The present invention relates to the field of molecular biology andbiotechnology. In particular, the invention relates to the field ofnucleic acid detection and identification. More in particular theinvention relates to the generation of a physical map of a genome, orpart thereof, using high-throughput sequencing technology.

BACKGROUND OF THE INVENTION

Integrated genetic and physical genome maps are extremely valuable formap-based gene isolation, comparative genome analysis and as sources ofsequence-ready clones for genome sequencing projects. The effect of theavailability of an integrated map of physical and genetic markers of aspecies for genome research is enormous. Integrated maps allow forprecise and rapid gene mapping and precise mapping of microsatelliteloci and SNP markers. Various methods have been developed for assemblingphysical maps of genomes of varying complexity. One of the bettercharacterized approaches use restriction enzymes to generate largenumbers of DNA fragments from genomic subclones (Brenner et al., Proc.Natl. Acad. Sci., (1989), 86, 8902-8906; Gregory et al., Genome Res.(1997), 7, 1162-1168; Marra et al., Genome Res. (1997), 7, 1072-1084).These fingerprints are compared to identify related clones and toassemble overlapping clones in contigs. The utility of fingerprintingfor ordering large insert clones of a complex genome is limited,however, due to variation in DNA migration from gel to gel, the presenceof repetitive DNAs, unusual distribution of restriction sites and skewedclone representation. Most high quality physical maps of complex genomeshave therefore been constructed using a combination of fingerprintingand PCR-based or hybridisation based methods. However, one of thedisadvantages of the use of fingerprinting technology is that it isbased on fragment-pattern matching, which is an indirect method.

It would be preferred to create physical maps by generating the contigsbased on actual sequence data, i.e. a more direct method. Asequence-based physical map is not only more accurate, but at the sametime also contributes to the determination of the whole genome sequenceof the species of interest. Recently methods for high throughputsequencing have been made available that would allow for thedetermination of complete nucleotide sequences of clones in a moreefficient and cost-effective manner.

However, detection by sequencing of the entire restriction fragment isstill relatively uneconomical. Furthermore, the current state of the artsequencing technology such as disclosed herein elsewhere (from 454 LifeSciences, www.454.com, Solexa, www.solexa.com, and Helicos,www.helicosbio.com), despite their overwhelming sequencing power, canonly provide sequencing fragments of limited length. Also the currentmethods do not allow for the simultaneous processing of many samples inone run.

It is now the goal of the present invention to devise and describe astrategy that allows for the high throughput generation of a physicalmap based on a combination of restriction digestion, pooling, highlyaccurate amplification and high throughput sequencing. Using thismethod, physical maps can be generated, even of complex genomes.

Definitions

In the following description and examples a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided. Unless otherwise defined herein,all technical and scientific terms used have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. The disclosures of all publications, patentapplications, patents and other references are incorporated herein intheir entirety by reference.

Nucleic acid: a nucleic acid according to the present invention mayinclude any polymer or oligomer of pyrimidine and purine bases,preferably cytosine, thymine, and uracil, and adenine and guanine,respectively (See Albert L. Lehninger, Principles of Biochemistry, at793-800 (Worth Pub. 1982) which is herein incorporated by reference inits entirety for all purposes). The present invention contemplates anydeoxyribonucleotide, ribonucleotide or peptide nucleic acid component,and any chemical variants thereof, such as methylated, hydroxymethylatedor glycosylated forms of these bases, and the like. The polymers oroligomers may be heterogenous or homogenous in composition, and may beisolated from naturally occurring sources or may be artificially orsynthetically produced. In addition, the nucleic acids may be DNA orRNA, or a mixture thereof, and may exist permanently or transitionallyin single-stranded or double-stranded form, including homoduplex,heteroduplex, and hybrid states.

AFLP: AFLP refers to a method for selective amplification of nucleicacids based on digesting a nucleic acid with one or more restrictionendonucleases to yield restriction fragments, ligating adaptors to therestriction fragments and amplifying the adaptor-ligated restrictionfragments with at least one primer that is (in part) complementary tothe adaptor, (in part) complementary to the remains of the restrictionendonuclease, and that further contains at least one randomly selectednucleotide from amongst A, C, T, or G (or U as the case may be). AFLPdoes not require any prior sequence information and can be performed onany starting DNA. In general, AFLP comprises the steps of:

-   -   (a) digesting a nucleic acid, in particular a DNA or cDNA, with        one or more specific restriction endonucleases, to fragment the        DNA into a corresponding series of restriction fragments;    -   (b) ligating the restriction fragments thus obtained with a        double-stranded synthetic oligonucleotide adaptor, one end of        which is compatible with one or both of the ends of the        restriction fragments, to thereby produce adaptor-ligated,        preferably tagged, restriction fragments of the starting DNA;    -   (c) contacting the adaptor-ligated, preferably tagged,        restriction fragments under hybridizing conditions with one or        more oligonucleotide primers that contain selective nucleotides        at their 3′-end;    -   (d) amplifying the adaptor-ligated, preferably tagged,        restriction fragment hybridised with the primers by PCR or a        similar technique so as to cause further elongation of the        hybridised primers along the restriction fragments of the        starting DNA to which the primers hybridised; and    -   (e) detecting, identifying or recovering the amplified or        elongated DNA fragment thus obtained.

AFLP thus provides a reproducible subset of adaptor-ligated fragments.AFLP is described in inter alia EP 534858, U.S. Pat. No. 6,045,994 andin Vos et al. (Nucleic Acid Research, 1995, 23, 21, 4407-4414) Referenceis made to these publications for further details regarding AFLP. TheAFLP is commonly used as a complexity reduction technique and a DNAfingerprinting technology. Within the context of the use of AFLP as afingerprinting technology, the concept of an AFLP marker has beendeveloped.

Selective base: located at the 3′ end of the primer that contains a partthat is complementary to the adaptor and a part that is complementary tothe remains of the restriction site, the selective base is randomlyselected from amongst A, C, T or G. By extending a primer with aselective base, the subsequent amplification will yield only areproducible subset of the adaptor- ligated restriction fragments, i.e.only the fragments that can be amplified using the primer carrying theselective base. Selective nucleotides can be added to the 3′ end of theprimer in a number varying between 1 and 10. Typically 1-4 suffice andare preferred. Both primers may contain a varying number of selectivebases. With each added selective base, the number of amplifiedadaptor-ligated restriction fragments (amplicons) in the subset isreduced by a factor of about 4. Typically, the number of selective basesused in AFLP is indicated by +N+M, wherein one primer carries Nselective nucleotides and the other primers carries M selectivenucleotides. Thus, an Eco/Mse+1/+2 AFLP is shorthand for the digestionof the starting DNA with EcoRI and MseI, ligation of appropriateadaptors and amplification with one primer directed to the EcoRIrestricted position carrying one selective base and the other primerdirected to the MseI restricted site carrying 2 selective nucleotides. Aprimer used in AFLP that carries at least one selective nucleotide atits 3′ end is also depicted as an AFLP-primer. Primers that do not carrya selective nucleotide at their 3′ end and which in fact arecomplementary to the adaptor and the remains of the restriction site aresometimes indicated as AFLP+0 primers.

Clustering: with the term “clustering” is meant the comparison of two ormore nucleotide sequences based on the presence of short or longstretches of identical or similar nucleotides and grouping together thesequences with a certain minimal level of sequence homology based on thepresence of short (or longer) stretches of identical or similarsequences.

Assembly: construction of a contig based on ordering a collection of(partly) overlapping sequences, also called “contig building”.

Alignment: positioning of multiple sequences in a tabular presentationto maximize the possibility for obtaining regions of sequence identityacross the various sequences in the alignment, e.g. by introducing gaps.Several methods for alignment of nucleotide sequences are known in theart, as will be further explained below.

Identifier: a short sequence that can be added an adaptor or a primer orincluded in its sequence or otherwise used as label to provide a uniqueidentifier. Such a sequence identifier (tag) can be a unique basesequence of varying but defined length uniquely used for identifying aspecific nucleic acid sample. For instance 4 by tags allow 4(exp4)=256different tags. Typical examples are ZIP sequences, known in the art ascommonly used tags for unique detection by hybridization (Iannone et al.Cytometry 39:131-140, 2000). Using such an identifier, the origin of aPCR sample can be determined upon further processing. In the case ofcombining processed products originating from different nucleic acidsamples, the different nucleic acid samples are generally identifiedusing different identifiers.

Sequencing: The term sequencing refers to determining the order ofnucleotides (base sequences) in a nucleic acid sample, e.g. DNA or RNA.

High-throughput screening: High-throughput screening, often abbreviatedas HTS, is a method for scientific experimentation especially relevantto the fields of biology and chemistry. Through a combination of modernrobotics and other specialised laboratory hardware, it allows aresearcher to effectively screen large amounts of samplessimultaneously.

Restriction endonuclease: a restriction endonuclease or restrictionenzyme is an enzyme that recognizes a specific nucleotide sequence(target site) in a double-stranded DNA molecule, and will cleave bothstrands of the DNA molecule at or near every target site.

Restriction fragments: the DNA molecules produced by digestion with arestriction endonuclease are referred to as restriction fragments. Anygiven genome (or nucleic acid, regardless of its origin) will bedigested by a particular restriction endonuclease into a discrete set ofrestriction fragments. The DNA fragments that result from restrictionendonuclease cleavage can be further used in a variety of techniques andcan for instance be detected by gel electrophoresis.

Ligation: the enzymatic reaction catalyzed by a ligase enzyme in whichtwo double-stranded DNA molecules are covalently joined together isreferred to as ligation. In general, both DNA strands are covalentlyjoined together, but it is also possible to prevent the ligation of oneof the two strands through chemical or enzymatic modification of one ofthe ends of the strands. In that case the covalent joining will occur inonly one of the two DNA strands.

Synthetic oligonucleotide: single-stranded DNA molecules havingpreferably from about 10 to about 50 bases, which can be synthesizedchemically are referred to as synthetic oligonucleotides. In general,these synthetic DNA molecules are designed to have a unique or desirednucleotide sequence, although it is possible to synthesize families ofmolecules having related sequences and which have different nucleotidecompositions at specific positions within the nucleotide sequence. Theterm synthetic oligonucleotide will be used to refer to DNA moleculeshaving a designed or desired nucleotide sequence.

Adaptors: short double-stranded DNA molecules with a limited number ofbase pairs, e.g. about 10 to about 50 base pairs in length, which aredesigned such that they can be ligated to the ends of restrictionfragments. Adaptors are generally composed of two syntheticoligonucleotides which have nucleotide sequences which are partiallycomplementary to each other. When mixing the two syntheticoligonucleotides in solution under appropriate conditions, they willanneal to each other forming a double-stranded structure. Afterannealing, one end of the adaptor molecule is designed such that it iscompatible with the end of a restriction fragment and can be ligatedthereto; the other end of the adaptor can be designed so that it cannotbe ligated, but this need not be the case (double ligated adaptors).

Adaptor-ligated restriction fragments: restriction fragments that havebeen capped by adaptors.

Primers: in general, the term primers refer to DNA strands which canprime the synthesis of DNA. DNA polymerase cannot synthesize DNA de novowithout primers: it can only extend an existing DNA strand in a reactionin which the complementary strand is used as a template to direct theorder of nucleotides to be assembled. We will refer to the syntheticoligonucleotide molecules which are used in a polymerase chain reaction(PCR) as primers.

DNA amplification: the term DNA amplification will be typically used todenote the in vitro synthesis of double-stranded DNA molecules usingPCR. It is noted that other amplification methods exist and they may beused in the present invention without departing from the gist.

SUMMARY OF THE INVENTION

The present inventors have found by using a combination of restrictionenzyme digestion of clones in a library, adapter-ligation, (selective)amplification, high-throughput sequencing and deconvolution of theresulting sequences results in contigs that can be used to assemblephysical maps, even of large and complex genomes.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the invention relates to a method for the generation of aphysical map of at least part of a genome comprising the steps of:

-   -   (a) providing a sample DNA;    -   (b) generating an artificial chromosome (BAC, YAC) clone bank        wherein each artificial chromosome clone contains part of the        sample DNA;    -   (c) combining the artificial chromosome clones in one or more        pools, wherein each clone is present in more than one pool, to        create a library;    -   (d) digesting the DNA of one or more pools with one or more        restriction endonucleases to provide for a set of restriction        fragments for each pool;    -   (e) ligating adaptors to one or both sides of the restriction        fragments, wherein at least one adaptor contains a pool-specific        identifier or a degenerate identifier section, respectively, to        provide adaptor-ligated restriction fragments;    -   (f) optionally, combining the adapto-ligated restriction        fragments;    -   (g) amplifying the adaptor-ligated restriction fragments of        step (e) with at least one primer, which primer contains a        pool-specific section corresponding to the pool-specific        identifier section in the adaptor or contains a pool-specific        identifier at the position of the degenerate identifier section,        respectively, to provide tagged amplified adaptor-ligated        restriction fragments (amplicons);    -   (h) optionally, combining the amplicons in a set of combined        amplicons;    -   (i) determining the sequence of at least the pool-specific        identifier and part of the restriction fragment of the amplicons        or set of combined amplicons;    -   (j) assigning the restriction fragment sequences determined in        the amplicons of step (i) to the corresponding clones using the        pool-specific identifiers;    -   (k) ordering the restriction fragments derived from the same        clone to build a contig;    -   (l) ordering the contigs of the clones of step (k) to thereby        build a clone-contig and generate a physical map.        In step (a) of the method a sample DNA is provided. This can be        achieved by any means in the art such as disclosed for instance        by Sambrook et al (Sambrook and Russell (2001) “Molecular        Cloning: A Laboratory Manual (3^(rd) edition), Cold Spring        Harbor Laboratory, Cold Spring Harbor Laboratory Press). The        sample DNA can be from any species, in particular from human,        plant or animal origin. It is possible to use only a part of a        genome, but that is not necessary as the present invention also        provides for methods to accommodate genomes of any size, for        instance through the creation of reproducible subsets via        selective amplification based on AFLP, as described herein        elsewhere. Thus typically, the present method uses the entire        genome.

In step (b) an artificial clone bank is generated. The library can be aBacterial Artificial Chromosome library (BAC) or based on yeast (YAC).Other libraries such as based on cosmids, PAC, TAC or MAC are alsopossible. Preferred is a BAC library. The library is preferably of ahigh quality and preferably is a high insert size genomic library. Thismeans that the individual BAC contains a large insert of the genomic DNAunder investigation (typically >125 kbp). The size of the preferredlarge insert is species-dependent. Throughout this application referenceis made to BACs as examples of artificial chromosomes. However, it isnoted that the present invention is not limited thereto and that otherartificial chromosomes can be used without departing from the gist ofthe invention. Preferably the libraries contain at least five genomeequivalents, more preferably at least 7, most preferably at least 8.Particularly preferred is at least 10. The higher the number of genomeequivalents in the library, the more reliable the resulting contigs andphysical map will be.

The individual clones in the library are pooled to form pools containinga multitude of artificial chromosomes or clones. The pooling may be thesimple combination of a number of individual clones into one sample (forexample, 100 clones into 10 pools, each containing 10 clones), but alsomore elaborate pooling strategies may be used. The distribution of theclones over the pools is preferably such that each clone is present inat least two or more of the pools. Preferably, the pools contain from 10to 10000 clones per pool, preferably from 100 to 1000, more preferablyfrom 250 to 750. It is observed that the number of clones per pool canvary widely, and this variation is related to, for instance, the size ofthe genome under investigation. Typically, the maximum size of a pool ora sub-pool is governed by the ability to uniquely identify a clone in apool by a set of identifiers. As will be further elaborated onhereinbelow, a typical range for a genome equivalent in a pool is in theorder of 0.2-0.3, and this may again vary per genome. The pools aregenerated based on pooling strategies well known in the art. The skilledman is capable selecting the optimal pooling strategy based on factorssuch as genome size etc. The resulting pooling strategy will depend onthe circumstances, and examples thereof are plate pooling, N-dimensionalpooling such as 2D-pooling, 3D-pooling, 6D-pooling or complex pooling.To facilitate handling of large numbers of pools, the pools may, ontheir turn, be combined in super-pools (i.e. super-pools are pools ofpools of clones) or divided into sub-pools, as is exemplified in theappending FIG. 1 where a 3D pooling is illustrated. Other examples ofpooling strategies and their deconvolution (i.e. the correctidentification of the individual clone in a library by detection of thepresence of an known associated indicator (i.e. label or identifier) ofthe clone in one or more pools or subpools) are for instance describedin U.S. Pat. No. 6,975,943 or in Klein et al. in Genome Research,(2000), 10, 798-807. The pooling strategy is preferably such that everyclone in the library is distributed such over the pools that a uniquecombination of pools is made for every clone. The result thereof is thata certain combination of (sub)pools uniquely identifies a clone.

The pools are digested with restriction endonucleases to yieldrestriction fragments. Each pool is preferably separately subjected toan endonuclease digest. Each pool is treated with the same (combinationof) endonuclease(s). In principle any restriction endonuclease can beused. Restriction endonucleases may be frequent cutters (4 or 5 cutters,such as MseI or PstI) or rare cutters (6 and more cutters such as EcoRI,HindIII). Typically, restriction endonucleases are selected such thatrestriction fragments are obtained that are, on average, present in anamount or have a certain length distribution that is adequate for thesubsequent steps. In certain embodiments, two or more restrictionendonucleases can be used and in certain embodiments, combinations ofrare and frequent cutters can be used. For large genomes the use of, forinstance, three or more restriction endonucleases can be usedadvantageously.

To one or both ends of the restriction fragments, adaptors are ligatedin step (e) to provide for adaptor-ligated restriction fragments.Typically, adaptors are synthetic oligonucleotides as defined hereinelsewhere. The adaptors used in the present invention preferably containan identifier section, in essence as defined herein elsewhere. Incertain embodiments, the adaptor contains a pool-specific identifier,i.e. for each pool, an adapter containing a unique identifier is usedthat unequivocally indicates the pool. In certain embodiments, theadaptor contains a degenerate identifier section which is used incombination with a primer containing a pool-specific identifier.

In certain embodiments, the adapter-ligated restriction fragments can becombined in larger groups, in particular when the adaptors contain apool-specific identifier. This combination in larger groups may aid inreducing the number of parallel amplifications of each set ofadapter-ligated restriction fragments obtained from a pool.

The adaptor-ligated restriction fragments can be amplified using a setof primers of which at least one primer contains a pool-specificidentifier at the position of the pool-specific or degenerate identifierin the adaptor. This embodiment also allows for the grouping ofadaptor-ligated restriction fragments prior to the amplification asoutlined above. In an alternative embodiment, each pool ofadaptor-ligated restriction fragments, wherein the adaptor contained adegenerate identifier section, is amplified separately using a set ofprimers of which at least one primer contains a pool specific section,thereby uniquely identifying the pool.

Either way, the result is a set of amplified adapter-ligated restrictionfragments, also depicted as amplicons, that are linked to the pool fromwhich they originate by the presence in the amplicon of thepool-specific identifier. In certain embodiments, sub-sets of ampliconsmay be created by selective amplification using primers carryingselective nucleotides at their 3′ end, essentially as described hereinelsewhere.

The amplicons may be combined in certain embodiments, in a set ofcombined amplicons or a so-called sequence library.

In step (i) of the method, the amplicons are subjected to sequencing,preferably high throughput sequencing as described herein below. Duringsequencing, at least part of the nucleotide sequence of the amplicons isdetermined. Preferably at least the sequence of the pool-specificidentifier and part of the restriction fragment of the amplicons isdetermined. Preferably, a sequence of at least 10 nucleotides of therestriction fragment is determined In certain embodiments, at least 11,12, 13, 14 or 15 nucleotides of the restriction fragment are determinedThe number of nucleotides that are to be determined minimally will be,again, genome dependent. For instance, in plants more repetitivesequences are present, hence longer sequences (25-30 bp) are to bedetermined. For instance, calculations on the known genome ofArabidopsis have shown that, when including a 6 by restriction site inthe sequencing step, about 20 by per restriction fragment needs to bedetermined. It is possible to determine the sequence of the entirerestriction fragment, but this is not an absolute necessity for contigbuilding of a BAC clone.

In the sequencing step, to provide for increased accuracy, the sequencelibrary may be sequenced with a coverage of at least 5. This means thatthe sequence is determined of at least 5 amplicons obtained from theamplification of one specific adaptor-ligated restriction fragment. Inother words: each restriction fragment is (statistically) sequenced atleast five times. Increased coverage is preferred as its improvesaccuracy further, so preferably coverage is at least 7, more preferablya least 10. Increased coverage is used to compensate for a phenomenonthat is known as ‘sampling variation’.

In the following step, the (partly) sequenced amplicons are correlatedto the corresponding clone, typically in silico by means of computerizedmethods. The amplicons are selected that contain identical sections ofnucleotides in the restriction fragment-derived part. Subsequently thedifferent pool-specific identifiers are identified that are present inthose amplicons. The combination of the different pool-specificidentifiers and hence the sequence of the restriction fragment can beuniquely assigned to a specific clone (a process described earlier as‘deconvolution’). For example, in the case of a 3D pooling strategy(X,Y,Z), each pool in the library is uniquely addressed by a combinationof 3 pool-specific identifiers. Each clone occurs more than once in thelibrary, so for each occurrence of a clone in the library, a combinationof 3 pool-specific identifiers can be made in combination with the samerestriction fragment-derived section. In other words: a restrictionfragment-derived section originating from a clone will be tagged with 3different identifiers. Unique restriction fragment-derived sections,when observed in combination with the 3 identifiers can be assigned to asingle BAC clone. This can be repeated for each amplicon that containsother unique sections of nucleotides in the restriction fragment-derivedpart. This process of deconvolution can be made easier by keeping thegenome equivalent per pool relatively low (<0.3, pref. 0.2), therebyreducing the chance that the same fragment is present twice in the samepool derived from different clones. An exemplary representation of thepooling concept is provided in FIG. 1. A sample

DNA is converted into BAC library. The BAC library is pooled in a set ofpools (M) (3 pools are shown, each containing about 0.3 GE,). Each poolis divided into (X+Y+Z) subpools (typically a stack ofmicrotiterplates).

The sequenced amplicons that are now linked to a particular clone in thelibrary are used in building a contig based on sequence matching of therestriction fragment derived sections. The contigs of each clone arethen aligned to generate a physical map.

The advantages of the present method reside inter alia in the improvedaccuracy for BAC contig building compared to conventional technology forBAC contig building. Furthermore, physical map building based onsequence information is more accurate, as it is a direct way of physicalmap construction and contributes to the determination of the genomesequence, and further contributes sequence information suitable for STSdevelopment and comparative mapping purposes.

The high throughput sequencing used in the present invention is a methodfor scientific experimentation especially relevant to the fields ofbiology and chemistry. Through a combination of modern robotics andother specialised laboratory hardware, it allows a researcher toeffectively screen large amounts of samples simultaneously.

It is preferred that the sequencing is performed using high-throughputsequencing methods, such as the methods disclosed in WO 03/004690, WO03/054142, WO 2004/069849, WO 2004/070005, WO 2004/070007, and WO2005/003375 (all in the name of 454 Life Sciences), by Seo et al. (2004)Proc. Natl. Acad. Sci. USA 101:5488-93, and technologies of Helicos,Solexa, US Genomics, etcetera, which are herein incorporated byreference.

454 Life Sciences Technology

In certain embodiments, it is preferred that sequencing is performedusing the apparatus and/or method disclosed in WO 03/004690, WO03/054142, WO 2004/069849, WO 2004/070005, WO 2004/070007, and WO2005/003375 (all in the name of 454 Life Sciences), which are hereinincorporated by reference. The technology described allows sequencing of20 to 40 million bases in a single run and is 100 times faster andcheaper than competing technology. The sequencing technology essentiallycontains 5 steps: 1) fragmentation of DNA and ligation of specificadaptors to create a library of single-stranded DNA (ssDNA); 2)annealing of ssDNA to beads, emulsification of the beads in water-in-oilmicroreactors and performing emulsion PCR to amplify the individualssDNA molecules on beads; 3) selection of /enrichment for beadscontaining amplified ssDNA molecules on their surface 4) deposition ofDNA carrying beads in a PicoTiter™Plate; and 5) simultaneous sequencingin 100,000 wells by generation of a pyrophosphate light signal. Themethod will be explained in more detail below.

In a preferred embodiment, the sequencing comprises the steps of:

-   -   a. annealing adapted fragments to beads, each bead being        annealed with a single adapted fragment;    -   b. emulsifying and amplifying the annealed fragments on the        beads in water-in-oil microreactors, each water-in-oil        microreactor comprising a single bead;    -   c. loading the beads in wells, each well comprising a single        bead; and generating a pyrophosphate signal.

In the first step (a), sequencing adaptors are ligated to fragmentswithin the combination library. Said sequencing adaptor includes atleast a region for annealing to a complementary oligonucleotide bound toa bead, a sequencing primer region and a PCR primer region. Thus,adapted fragments are obtained.

In the first step, adapted fragments are annealed to the beads, eachbead annealing with a single adapted fragment. To the pool of adaptedfragments, beads are added in excess as to ensure annealing of onesingle adapted fragment per bead for the majority of the beads (Poissondistribution). In the present invention, the adapters that are ligatedto the restriction fragments obtained from the clones may comprise asection that is capable of annealing to a bead.

In a next step, the beads are emulsified in water-in-oil microreactors,each water-in-oil microreactor comprising a single bead. PCR reagentsare present in the water-in-oil microreactors allowing a PCR reaction totake place within the microreactors. Subsequently, the microreactors arebroken, and the beads comprising DNA (DNA positive beads) are enriched,i.e. separated from beads not containing amplified fragments.

In a following step, the enriched beads are loaded in wells, each wellcomprising a single bead. The wells are preferably part of aPicoTiter™Plate allowing for simultaneous sequencing of a large numberof fragments. After addition of enzyme-carrying beads, the sequence ofthe fragments is determined using pyrosequencing. In successive steps,the PicoTiter™Plate and the beads as well as the enzyme beads thereinare subjected to different deoxyribonucleotides in the presence ofconventional sequencing reagents, and upon incorporation of adeoxyribonucleotide a light signal is generated which is recorded.Incorporation of the correct nucleotide will generate a pyrosequencingsignal which can be detected.

Pyrosequencing itself is known in the art and described inter alia onwww.biotagebio.com; www.pyrosequencing.com/section technology. Thetechnology is further applied in e.g. WO 03/004690, WO 03/054142, WO2004/069849, WO 2004/070005, WO 2004/070007, and WO 2005/003375 (all inthe name of 454 Life Sciences), and Margulieset al., nature 2005, 437,376-380, which are herein incorporated by reference.

In the present invention, the beads are preferably equipped with primersequences or parts thereof that are capable of being extended bypolymerisation to yield bead-bound amplicons. In other embodiments, theprimers used in the amplification are equipped with sequences, forinstance at their 5′-end, that allow binding of the amplicons to thebeads in order to allow subsequent emulsion polymerisation followed bysequencing. Alternatively, the amplicons may be ligated with sequencingadaptors prior to ligation to the beads or the surface. The sequencedamplicons will reveal the identity of the identifier and hence thecombination of identifiers reveals the identity of the clone.

Solexa Technologies

One of the methods for high throughput sequencing is available fromSolexa, United Kingdom (www.solexa.co.uk) and described inter alia inWO0006770, WO0027521, WO0058507, WO123610, WO0157248, WO0157249,WO02061127, WO03016565, WO03048387, WO2004018497, WO2004018493,WO2004050915, WO2004076692, WO2005021786, WO2005047301, WO2005065814,WO2005068656, WO2005068089, WO2005078130. In essence, the method startswith adaptor-ligated fragments of DNA, in this particular case ofadapter-ligated restriction fragments of the artificial chromosome poolsas described herein elsewhere. The adaptor-ligated DNA is randomlyattached to a dense lawn of primers that are attached to a solidsurface, typically in a flow cell. The other end of the adaptor ligatedfragment hybridizes to a complementary primer on the surface. Theprimers are extended in the presence of nucleotides and polymerases in aso-called solid-phase bridge amplification to provide double strandedfragments. This solid phase bridge amplification may be a selectiveamplification. Denaturation and repetition of the solid-phase bridgeamplification results in dense clusters of amplified fragmentsdistributed over the surface. The sequencing is initiated by adding fourdifferently labelled reversible terminator nucleotides, primers andpolymerase to the flow cell. After the first round of primer extension,the labels are detected, the identity of the first incorporated bases isrecorded and the blocked 3′ terminus and the fluorophore are removedfrom the incorporated base. Then the identity of the second base isdetermined in the same way and so sequencing continues.

In the present invention, the adaptor-ligated restriction fragments orthe amplicons are bound to the surface via the primer binding sequenceor the primer sequence. The sequence is determined as outlined,including the identifier sequence and (part of) the restrictionfragment. Currently available Solexa technology allows for thesequencing of fragments of about 25 base pairs. By economical design ofthe adaptors and the surface bound primers, the sequencing step readsthrough the sample identifier, the remains of the recognition sequenceof the restriction endonuclease and any optional selective bases. When a6 by sample identifier is used, the remains are from the rare cutterEcoRI (AACCT), the use of two selective bases yields an internalsequence of the restriction fragment of 12 by that can be used touniquely identify the restriction fragment in the sample.

In a preferred embodiment based on the Solexa sequencing technologyabove, the amplification of the adapter ligated restriction fragments isperformed with a primer that contains at most one selective nucleotideat its 3′end, preferably no selective nucleotides at is 3′ end, i.e. theprimer is only complementary to the adaptor (a +0 primer).

In alternative embodiments directed to the sequencing methods describedherein, the primers used in the amplification may contain specificsections (as alternative to the herein described primer or primerbinding sequences) that are used in the subsequent sequencing step tobind the adaptor-capped restriction fragments or amplicons to thesurface. These are generally depicted as the key region or the 5′-primercompatible sequence.

The present invention further embodies itself in adaptors containingpool-specific or degenerated identifier sections and/or in primerscontaining pool-specific identifiers, respectively.

DESCRIPTION OF THE FIGURES

FIG. 1: Schematic representation of pooling strategies.

FIG. 2: Four continous BAC-contigs on Arabidopsis chromosome 4 -poolingstrategy

FIG. 3: No overlaps within the group, alternating minimal tiling path

FIG. 4: recognition sequence adressed BAC pools—amplified product onagarose-gel

FIG. 5: Re-assembled minimal tiling path-part of the 1.9 Mb contigenlarged

EXAMPLES De Novo BAC-Based Physical Map Construction of ArabidopsisThaliana Based on a Sequencing By Synthesis (SBS) Approach

This example is based on the following generalisations.

The total Arabidopsis thaliana genome is ˜125 Mbp. A BacterialArtificial Chromosome (BAC) has a genomic insert of ˜100 kb on average.One Genome Equivalent (GE) of BACs for a 1× physical coverage of theArabidopsis genome comprises ˜1250 BACs. For optimal results, it ispreferred that the construction of the BAC pools is such that one BACpool contains not more than 0.34 GE (˜384 BACs). Statistical analysispredicts that in 0.34 GE the chance of finding 2 identical BACs (that is2 BACs that would map to the exact same physical position) is <5%. LowerGE' in a BAC pool further reduces the chance of finding two BACs mappingto the same position. A straightforward 3D-pooling system is used forthe calculations. A total of 10 GE of BACs of 2 different high qualityBAC libraries (2 different cloning enzymes eg. EcoRI and HindIII) aresufficient for the construction of a high quality physical map. 10 GEBACs for Arabidopsis is ˜12.500 BACs.

The sequence Tags (the combination of part of the restriction fragmentand identifier) are generated from a rare cutter restriction site, forexample AFLP fragments such as EcoRI/MseI, or HindIII/MseI or acombination of several enzyme combinations (ECs).

In this example the enzyme combination HindIII/MseI is used. Thedistribution of HindIII/MseI fragments in the Arabidopsis genome isestimated to be between 50 to 120 fragments per 100 kb.

Set up for high throughput sequencing:

See also FIG. 1. 0.3 GE corresponds to 384 BACs. 3D-pooling of 384 BACs,with dimensions X+Y+Z results in 8+12+4=24 subpools. For 10 GE : M(X+Y+Z)=30 (8+12+4)=720 subpools.

For each subpool, the aim is to generate:

-   -   100 sequenced Tags per BAC    -   10 fold sequence redundancy per Tag    -   3 dimensional pooling (each BAC fragment is sequenced in each        (X,Y,Z) dimension)

This means that for bridging amplification-based high throughputsequencing of a pool of 0.34GE, a set of sequencing reads of: 8subpools×(12×4×100×10)+12 subpools×(8×4×100×10)+4subpools×(12×8×100×10)=1.152.000 reads are needed. This means for one GEthat 3*1.152.000=3.456.000 reads per GE are needed and 10×3.456.000reads per 10 GE=34.560.000 reads.

A single BAC generates a potential of ˜100 unique sequence tags of ˜20bps (including the restriction site). The number of sequences willdepend on the choice and/or combination of enzyme combinations.

The individual BAC coordinates and accompanying sequence tags can bededuced from the addressed subpool sequences by the “deconvolution”step. Consequently, via deconvolution each sequence tag is assignable tothe corresponding individual BAC. Repetitive sequence tags are ignored.The deconvolution process will result in a string of 100 Tags per BACs,and subsequently the assembly of a de novo physical map is achievedthrough a FPC (FingerPrintedContigs) type process, as described by CariSoderlund for BAC fragments analysed in agarose gels (Soderlund et al.2000—Genome Research 10; 1772-1787). Finally, the anchoring of thephysical map to the genetic map is performed in silico. For largergenomes other pooling strategies may be necessary.

De Novo BAC-Based Physical Map Construction of Cucumis Sativus Based ona Sequencing By Synthesis (SBS) Approach

This example is based on the following generalisations.

The total Cucumis sativus genome is ˜350 Mbp. A Bacterial ArtificialChromosome (BAC) has a genomic insert of ˜100 kb on average. One GenomeEquivalent (GE) of BACs for a 1× physical coverage of the Arabidopsisgenome comprises ˜3500 BACs. For optimal results, it is preferred thatthe construction of the BAC pools is such that one BAC pool contains notmore than 0.34 GE (˜384 BACs). Statistical analysis predicts that in0.34 GE the chance of finding 2 identical BACs (that is 2 BACs thatwould map to the exact same physical position) is <5%. Lower GE' in aBAC pool further reduces the chance of finding two BACs mapping to thesame position. A straightforward 3D-pooling system is used for thecalculations. A total of 10 GE of BACs of 2 different high quality BAClibraries (2 different cloning enzymes eg. EcoRI and HindIII) aresufficient for the construction of a high quality physical map. 10 GEBACs for Cucumis is 35.000 BACs.

The sequence Tags (the combination of part of the restriction fragmentand identifier) are generated from a rare cutter restriction site, forexample AFLP fragments such as EcoRI/MseI, or HindIII/MseI or acombination of several enzyme combinations (ECs).

In this example the enzyme combination HindIII/MseI is used. Thedistribution of HindIII/MseI fragments in the Cucumis sativus genome isestimated to be between 50 to 120 fragments per 100 kb.

Set up for high throughput sequencing:

See also FIG. 1. 0.3 GE corresponds to 1152 BACs. 3D-pooling of 1152BACs, with dimensions X+Y+Z results in 8+12+12=32 subpools. For 10 GE :M (X+Y+Z)=30(8+12+12)=960 subpools.

For each subpool, the aim is to generate:

-   -   100 sequenced Tags per BAC    -   10 fold sequence redundancy per Tag    -   3 dimensional pooling (each BAC fragment is sequenced in each        (X,Y,Z) dimension)

This means that for bridging amplification-based high throughputsequencing of a pool of 0.34GE, a set of sequencing reads of: 8subpools×(12×12×100×10)+12 subpools×(8×12×100×10)+12subpools×(12×8×100×10)=3.456.000 reads are needed. This means for one GEthat 3*3.456.000=10.368.000 reads per GE are needed and 10×10.368.000reads per 10 GE=103.680.000 reads.

A single BAC generates a potential of ˜100 unique sequence tags of ˜20bps (including the restriction site). The number of sequences willdepend on the choice and/or combination of enzyme combinations.

The individual BAC coordinates and accompanying sequence tags can bededuced from the addressed subpool sequences by the “deconvolution”step. Consequently, via deconvolution each sequence tag is assignable tothe corresponding individual BAC. Repetitive sequence tags are ignored.The deconvolution process will result in a string of 100 Tags per BACs,and subsequently the assembly of a de novo physical map is achievedthrough a FPC (FingerPrintedContigs) type process, as described by CariSoderlund for BAC fragments analysed in agarose gels (Soderlund et al.2000—Genome Research 10; 1772-1787). Finally, the anchoring of thephysical map to the genetic map is performed in silico. For largergenomes other pooling strategies may be necessary.

AFLP templates (EcoRI/MseI or HindIII/MseI) are prepared from pooledBACs. AFLP amplification is performed using a combination 2 HindIII+1primers and an MseI+0 primer (same for EcoRI). The use of two +1 primerensures amplification of approximately 50% of the H/M (or E/M) fragmentsfrom the pools, i.e. on average 70/2=35 restriction fragments areamplified for each enzyme combination. The AFLP amplification reactionsare performed with AFLP primers containing unique identifier tags at the5′end for each of the BAC pools. Hence at least 74 identifier sequencesare needed. This can be accomplished with 4 base tags (4⁴=256possibilities). Identifier sequences are only needed for the HindIIIprimer, since unidirectional sequencing will be performed in thisexample.

AFLP reaction mixtures of all pools are mixed in equal amounts, creatinga fragment library. The fragment library is used to construct a sequencelibrary.

Given a 3-D pooling strategy, this means that every fragment is sampleda plurality of times on average in each dimension. Results are 100 bysequences derived from the HindIII (or EcoRI) site of the restrictionfragments. As said, per BAC clone an average of 35 sequences areobtained. The sequences form the basis for contig assembly using aprocedure similar to FPC (Software package by Soderlund obtainable fromhttp:www.agcol.arizona.edu/software/fpc/) but based on sequence matching(more detailed).

The advantage of the use of reproducible complexity reduction is thatless fragments are needed for the construction of a physical map. Acomplexity reduction of 50% in the above Cucumis example leads to51.840.000 reads instead of 103.680.000. A further advantage of thepresent invention is, using complexity reduction as described herein,that physical maps can be generated of controllable quality. This meansthat by reducing a BAC pool in complexity by a +1 AFLP amplification,for instance a primer combination with +C, results in a physical map ofabout 25% of the quality (coverage) compared to a +1 amplification withall four primer combinations (A, C, T, G). However, when two or threeprimer combinations are used, increased coverage is obtained, i.e. forinstance 55% or 90%, respectively, compared to the coverage obtainedwith a +1 amplification with all four primer combinations (A, C, T, G).

BAC Clones Addresses:

Fragments derived from the same BAC clone are amplified with 3 differenttagged primers. Hence, unique sequences observed in combination with 3tags are assigned to a single BAC clone in the library. Repeatedsequences are observed in combinations with multiple tags and cantherefore not be connected to a single BAC clone. This affects aconsiderable proportion of the fragments, but among 35 fragments/BACclone, at a least a subset is unique.

A 10-fold sequence coverage of the BAC pools (3.3 fold/dimension) meansthat not all expected fragments are observed (due to concentrationdifferences of individual clones and sampling variation etc). Hence afraction of the (unique) sequences is only observed in combination with1 or 2 tags (or not at all), which precludes assigning them to a singleBAC clone. However, to the extent that this is due to sampling variationbetween the restriction fragments derived from the same clone, the factthat 35 fragments are sampled means that the combination of tagsprovides the correct address for the BAC: see below.

Tag 1 Tag 2 Tag 3 Fragment 1 X X Fragment 2 X X Fragment 3 X X Fragment4 X X Fragment 5 X X X Etc. Fragment 35 X X

The scheme above illustrates that contig building groups the fragmentstogether in a contig; fragment 5, which has a unique sequence and wassampled in combination with 3 tags defines the address of the BAC inlibrary, from which fragment 1-4 (+35) are probably derived as well.

Hence, the strength of the approach is that sequence information on asufficiently large number of restriction fragments (35 in the aboveexample) is used to build accurate contigs, while the use of a 3dimensional tagging system allow direct identification for the majorityof BACs, even though the BAC address can not be derived from eachindividual fragment sequence (due to experimental variation). However,the combination of tags from fragments derived from the same BAC willprovide the BAC address.

Thus, the information derived from sequence-based BAC contiging is thesame as for conventional approaches (i.e. contig+BAC address). It isobserved that for individual clone fingerprinting approaches, the BACaddress will be known by definition.

Example 2

Procedure for High Throughput Physical Mapping by Sequence tag BACMapping.

A total of 72 BACs (BAC=Bacterial Artificial Chromosome) mapping tochromosome 4 of Arabidopsis and spanning a total physical stretch of 5.4Mb in 4 BAC contigs (1.8 Mb, 1.2 Mb, 0.5 Mb and 1.9 Mb) were selectedfrom the TAIR and other databases. The donor plant of the BAC librariesis Arabidopsis thaliana ecotype Colombia. The 72 BACs, ranging in sizebetween 70 kb and 150 kb, were separated in 2 groups of 36 BACs, group“AB” and group “XY”. Within the 2 groups the 36 BACs have no internaloverlap, while the BACs of group AB and group XY combined can beassembled into 4 continuous minimal tiling path contigs with alternatingBACs from group AB and XY (see FIGS. 2-5).

Pooling Strategy for 72 Arabidopsis BACs, 36 in Group AB and 36 in GroupXY

GroupAB

B1 B2 B3 B4 B5 B6 A1 F23J03 T30A10 T25P22 T09A04 T05L19 F07L13 A2 T12H20T22B04 F25E04 T26M18 T04C09 F07K02 A3 F07K19 F16G20 T32A16 T22A06 F06i07F24A06 A4 F08F16 F28M20 F10N07 F08B04 T16i18 F04i10 A5 T16L01 F17i05F28A23 T04L20 T12J05 F23E12 A6 F14H08 T19P05 T10C14 F06D23 T03E09 T06O13

GroupXY

Y1 Y2 Y3 Y4 Y5 Y6 X1 T03H13 T08A17 T15G18 F17A08 F28M11 F24G24 X2 T04F09F25i24 F08L21 T05C23 F16J13 T01P17 X3 T12H17 F21P08 F09D16 T19F09 F22K18F13M23 X4 T30C03 F03L17 F11C18 F10M06 F04D11 F26P21 X5 F17M05 T09O24T04G07 F10M10 F11i11 T04K12 X6 F05M05 T19K04 F23E13 T02G10 F07O06 T08H13T = TAMU BAC library - 12.5 microgram chloramphenicol/ml F = IGF BAClibrary - 50 microgram kanamycine/ml

The 72 BACs were grown overnight as individual clones in 200 microliterstandard TY medium including chloramphenicol (TAMU BAC clones) orkanamycine (IGF BAC clones). All clones were grown in a 6×6 format tofacilitate the pooling procedure. In the morning the liquid culture waspooled in 2 dimensions (6×6) such that 12 pools per group weregenerated. Each pool contained 600 microliter of medium with grown BACs(100 microliter per individual BAC). DNA was isolated from all 24 BACpools following a standard alkaline miniprep procedure according toSambrook et al. (2001).

50 ng DNA of each BAC pool was digested with restriction enzymes EcoRIand MseI, and subsequently EcoRI and MseI AFLP adaptors were ligated,according to the standard AFLP procedure described by Vos et al. (1995).The restriction/ligation mix was diluted 10× in MilliQ-water and 5microliter was used in the amplification step. The primers used in theamplification step were designed with a 4 nucleotide recognitionsequence, such that each pool is tagged with a pool specific 4nucleotide address-sequence. This recognition sequence is necessary tofacilitate the deconvolution of all sequences to an individualBAC-coordinate.

Both the EcoRI+0 and MseI+0 primers used were adapter compatible5′-phosphorylated primers carrying 5′-recognition sequences and aredifferent for each pool coordinate (see FIG. 4). The 5′-phosphorylationis necessary for the ligation of the pyrosequencing adaptors.Amplification was performed for 30 cycli with the profile: 94° C. (30sec), 56° C. (60 sec), 72° C. (60 sec). After amplification the productswere checked on agarose gel (FIG. 4) and the 12 amplified pool-productsof each group were pooled into a group-pool (AB cq. XY) and quantified.Five micrograms DNA of each group-pool was immediately processed in thefurther preparation steps for 454 sequencing. 454 pyrosequencing wasperformed on the GS20 platform according to Margulies et al. (2005).

Analysis of the Dataset and Assembly of the BAC Contigs

The list of DNA sequence reads as generated by the GS20 pyrosequencingmachine were parsed in 3 steps:

Step 1) the first 4 nucleotides consisting of the pool sample code wereidentified and the corresponding pool-labels were assigned. If the codewas unknown, the read was removed from the set.

Step 2) the next 16 or 17 nucleotides (depending on the restrictionenzyme) containing the primer sequence were identified. When 100%identical to the primer sequence the reads were approved and added tothe dataset and otherwise removed.

Step 3) all reads from step 2 were trimmed to 14 nucleotides after theprimer sequence.

All correct trimmed sequence reads were subsequently grouped: all 100%identical reads were identified and assigned to their correspondingpool. Each unique group of reads is termed a ‘tag’. Tags that were foundin exactly 2 pools, both one for the X-coordinate and one for theY-coordinate, were linked to a specific BAC: this procedure is calleddeconvolution.

All unique tags for deconvolved BACs were listed for both BAC groups.Pairs of BACs with one or more common tags were identified. Subsequentlythe BAC contigs could be assembled as shown in table 1.

TABLE 1 BAC links from all sequence tags, common between pairs of BACs(e.g. X1Y1 and A1B1) and occurring at least 2 times in each pool.Contigs are numbered. BAC link NrTags Contig X1Y1_A1B1 8 Contig1X1Y2_A1B1 18 Contig1 X1Y2_A1B2 6 Contig1 X1Y3_A1B2 19 Contig1 X1Y3_A1B33 Contig1 X1Y4_A1B3 10 Contig1 X1Y4_A1B5 10 Contig1 X1Y5_A1B4 16 Contig1X1Y5_A1B5 12 Contig1 X1Y6_A1B4 13 Contig1 X1Y6_A1B6 4 Contig1 X2Y1_A1B61 Contig1 X2Y1_A2B1 3 Contig1 X2Y2_A2B1 4 Contig1 X2Y2_A2B2 2 Contig1X2Y3_A2B2 5 Contig1 X2Y4_A2B3 4 Contig2 X2Y4_A2B4 2 Contig2 X2Y5_A2B4 1Contig2 X2Y5_A2B5 1 Contig2 X2Y6_A2B5 4 Contig2 X3Y1_A2B6 3 Contig3X3Y1_A3B1 5 Contig3 X3Y2_A3B1 4 Contig3 X3Y2_A3B2 2 Contig3 X3Y3_A3B2 1Contig3 X3Y3_A3B3 5 Contig3 X3Y4_A3B3 15 Contig3 X3Y4_A3B4 1 Contig3X3Y5_A3B4 2 Contig3 X3Y5_A3B5 13 Contig3 X3Y6_A3B5 7 Contig3 X3Y6_A3B6 7Contig3 X4Y1_A3B6 10 Contig3 X4Y2_A4B1 12 Contig4 X4Y2_A4B2 4 Contig4X4Y3_A4B2 5 Contig4 X4Y3_A4B3 20 Contig4 X4Y4_A4B3 5 Contig4 X4Y4_A4B411 Contig4 X4Y5_A4B5 9 Contig5 X4Y6_A4B5 7 Contig5 X4Y6_A4B6 6 Contig5X5Y1_A5B1 6 Contig6 X5Y2_A5B1 5 Contig6 X5Y2_A5B2 28 Contig6 X5Y3_A5B2 4Contig6 X5Y3_A5B3 26 Contig6 X5Y4_A5B4 4 Contig7 X5Y5_A5B4 3 Contig7X5Y5_A5B5 1 Contig7 X5Y6_A5B5 16 Contig7 X5Y6_A5B6 19 Contig7 X6Y1_A5B67 Contig7 X6Y1_A6B1 14 Contig7 X6Y2_A6B1 3 Contig7 X6Y2_A6B2 14 Contig7X6Y3_A6B2 14 Contig7 X6Y3_A6B3 8 Contig7 X6Y4_A6B3 14 Contig7 X6Y5_A6B513 Contig8 X6Y6_A6B5 8 Contig8 X6Y6_A6B6 14 Contig8

It was demonstrated that the 4 BAC minimal tiling paths of 1.8 Mb, 1.2Mb, 0.5 Mb and 1.9 Mb could be reassembled in a straightforward wayafter the deconvolution of sequence tags to the individual BACs (table 1and FIG. 4). A comparison of the generated GS20 tags with predictedfragments in silico showed that 70 to 80% of the EcoRI/MseI fragmentswere sequenced. Therefore in the reassembly of the 4 BAC contigs some ofthe smaller physical overlaps between 2 BACs could not be detected.

The fact that short reads (14 bp) are sufficient to reassemble the BACtiling paths indicates that high throughput sequencing platforms withshort read length (such as the Illumina Genome Analyser and SOliD (ABI))enables high throughput physical map assembly following the proposedmethod.

1.-20. (canceled)
 21. Method for the generation of a physical map of atleast part of a genome comprising the steps of: (a) providing a sampleDNA; (b) generating an artificial chromosome clone bank wherein eachartificial chromosome clone contains part of the sample DNA; (c)combining the artificial chromosome clones in a plurality of pools,wherein each clone is present in more than one pool of the plurality ofpools, to create a library; (d) digesting the one or more pools with oneor more restriction endonucleases to provide for a set of restrictionfragments for each pool; (e) ligating adaptors to one or both sides ofthe restriction fragments, wherein at least one adaptor contains apool-specific identifier or a degenerate identifier section,respectively, to provide adaptor-ligated restriction fragments; (f)amplifying the adaptor-ligated fragments with at least one primer toprovide amplicons; (g) determining the sequence of at least thepool-specific identifier and part of the restriction fragment of theamplicons; (h) assigning the restriction fragment sequences determinedin the amplicons to the corresponding clones using the pool-specificidentifiers; (i) building a contig based on sequence matching of therestriction fragment derived sections; (j) ordering the restrictionfragments of step (i) to thereby build a clone-contig and generating aphysical map.
 22. Method according to claim 21, wherein theadaptor-ligated restriction fragments of step (e) are combined. 23.Method according to claim 22, wherein the primer contains apool-specific section corresponding to the pool-specific identifiersection in the adaptor or contains a pool-specific identifier at theposition of the degenerate identifier section, respectively, to provideamplified adaptor-ligated restriction fragments (amplicons).
 24. Methodaccording to claim 23, wherein the amplicons are combined in a set ofcombined amplicons.
 25. Method according to claim 21, wherein therestriction fragments are assigned to the corresponding clone byclustering adapter-ligated restriction fragments or amplicons thatcontain identical sequences in (part of the) restriction fragments butcarry different pool-specific identifiers.
 26. Method according to claim21, wherein the sequencing is carried out by means of high-throughputsequencing.
 27. Method according to claim 26, wherein thehigh-throughput sequencing is performed on a solid support.
 28. Methodaccording to claim 26, wherein the high-throughput sequencing is basedon Sequencing-by-Synthesis.
 29. Method according to claim 26, whereinthe high-throughput sequencing comprises the steps of: annealing theamplicons or adapter-ligated restriction fragments to beads, each beadannealing with a single adapter-ligated restriction fragments oramplicon; emulsifying the beads in water-in-oil micro reactors, eachwater-in-oil micro reactor comprising a single bead; performing emulsionPCR to amplify adapter-ligated restriction fragments or amplicons on thesurface of heads; optionally, selecting/enriching beads containingamplified amplicons; loading the beads in wells, each well comprising asingle bead; and generating a pyrophosphate signal.
 30. Method accordingto claim 26, wherein the high-throughput sequencing comprises the stepsof: annealing the adapter-ligated restriction fragments or amplicons toa surface containing first and second primers or first and second primerbinding sequences respectively, performing bridge amplification toprovide clusters of amplified adapter-ligated restriction fragments oramplified amplicons, determining the nucleotide sequence of theamplified adapter-ligated restriction fragments or amplified ampliconsusing labelled reversible terminator nucleotides.
 31. Method accordingto claim 21, wherein the identifier is from 4-16 bp.
 32. Methodaccording to claim 31, wherein the identifier is from 4-10 bp. 33.Method according to claim 32, wherein the identifier is from 4-8 bp. 34.Method according to claim 33, wherein the identifier is from 4-6 bp. 35.Method according to claim 31, wherein the identifier does not contain 2or more identical consecutive bases.
 36. Method according to claim 31,wherein for two or more clones, the corresponding identifiers contain atleast two different nucleotides.
 37. Method according to claim 21,wherein at least one primer carries 1-10 selective nucleotides at its 3′end to provide for a subset of amplicons.
 38. Method according to claim37, wherein the at least one primer carries 1- 4 selective nucleotidesat it 3′ end.